Method for Irradiating Thrombocyte Concentrates in Flexible Containers With Ultra-Violet Light

ABSTRACT

The invention relates to a method for inactivating pathogens such as bacteria and viruses and/or leucocytes in thrombocyte concentrations by irradiation in flexible containers with ultra-violet light using agitation. The blood product is packaged in a flexible bag to permit the intermixing of the fluid by agitation (tilting, rotation, translation). This is also promoted by the filling of the bag to a maximum 30% of the filling capacity.

The subject matter of the invention is a method for inactivatingpathogens such as bacteria and viruses and/or leucocytes in plateletconcentrates (thrombocyte concentrates, PCs) by irradiation withultraviolet light.

It is known that the therapeutic application of blood preparationscarries the risk that the recipients of the blood preparation areinfected with viruses and bacteria. The viruses hepatitis B (HBV) andhepatitis C (HCV) and the Aids pathogens HIV 1 and HIV 2 are named byway of example. The risk always exists when, during the production ofthe preparation, no steps are taken to inactivate or eliminate the saidpathogens.

Ultraviolet (UV) light is differentiated according to wavelength. In thecontext of this application, the following definition is applied: UVA:less than 400 to 320 nm, UVB: less than 320 to 280 nm and UVC less than280 to 200 nm. It is known that by irradiation with short-waveultraviolet (UV) light, i.e. in the wave length range belowapproximately 320 nm (UVB and UVC), viruses and also bacteria can beinactivated, for instance in blood plasma or in cellular bloodpreparations. Above 320 nm, the energy of the radiation is too low toinactivate microorganisms and viruses. Compared with chemical,photochemical and photodynamic methods of pathogen inactivation, mereirradiation with UV light essentially has the advantage of beingeffective on its own and not requiring the addition of reactivechemicals or photoactive substances.

Such additives or their split products or photo products frequentlyrequire subsequent removal, because they are toxic or mutagenic. Inaddition, they can cause the formation of neoantigenic structures in thetreated preparation when they bind to plasma proteins and cell surfaces.Generally, such additives are not able to be removed completely; atleast, their removal requires additional effort. Such additional workingsteps can, moreover, impair the quality of the sterilised preparations.UVC is the most effective for direct pathogen inactivation. However, ithas the disadvantage that it only penetrates protein-containingsolutions such as blood plasma or turbid suspensions (e.g. PCs) up to avery small penetration depth.

UVC was used during the Second World War and also shortly thereafter tosterilise blood plasma and albumin solutions, especially in order toinactivate hepatitis viruses. At that time, one proceeded so that thesolution was directed in a through-flow apparatus as a thin film past aUVC light source. The method proved to be not sufficiently reliable andwas abandoned (Kallenbach N R, Cornelius P A, Negus D, et al.Inactivation of viruses by ultraviolet light. Curr Stud Hematol BloodTranfus 1989, 56, 70-82).

In the present day, methods are used which have been further developed,operating according to the same principle, in order to sterilisetherapeutic plasma protein preparations. In all cases the concern was oris with treating larger volumes, i.e. plasma pools or protein solutionsup to several hundred litres and even more (Hart H, Reid K, Hart W.Inactivation of viruses during ultraviolet light treatment of humanintravenous immunoglobulin and albumin. Vox Sang 1993; 64(2):82-8, andChin S, Williams B, Gottlieb P, et al. Virucidal short wavelengthultraviolet light treatment of plasma and factor VIII concentrate:protection of proteins by antioxidants; Blood 1995; 86(11):4331-6).

For sterilising a plurality of individual units of PCs, which have beenobtained from blood donations or by mechanical apheresis—with a volumeof at most up to several hundred ml—the aforementioned through-flowapparatus are not suitable. However, this is in fact necessary in thedaily practice of a blood bank.

UVB is likewise microbiocidal and virucidal, although not to the sameextent as UVC. It penetrates protein-containing solutions and turbidsuspensions somewhat better than UVC, but its penetration depth, e.g. inplasma or PCs, can also only be established in the range of a fewmillimetres. Irradiation with UVB was tested, in order to inactivateT-lymphocytes in PCs, which are considerably more UV-sensitive thanviruses or bacteria. Hereby, an allo-immunisation against exogenous HLAantigens in the recipients of the preparations is to be prevented, whichcan cause the recipients to become refractory against furthertransfusions of PCs (Andreu G, Boccaccio C, Lecrubier C, et al.Ultraviolet irradiation of platelet concentrates: feasibility intransfusion practice. Transfusion 1990; 30(5):401-6 and Pamphilon D H.The rationale and use of platelet concentrates irradiated withultraviolet-B light. Transfus Med Rev 1999; 13(4):323-33).

In fact, the method did not win through, because the leucocytefiltration, which was developed at almost the same time, constitutes analternative which is similarly effective but is more favourable from thepoint of view of cost and effort (Leukocyte reduction and ultraviolet Birradiation of platelets to prevent alloimmunization and refractorinessto platelet transfusions. The Trial to Reduce Alloimmunization toPlatelets Study Group. N Engl J Med 1997; 337(26):1861-9).

It was likewise described that viruses in platelet suspensions can beinactivated by irradiation with monochromatic UVB light (wavelength 308nm). An excimer laser was used here; the test volume was a few ml(Prodouz K N, Fratantoni J C, Boone E J, Bonner R F. Use of laser-UV forinactivation of virus in blood products. Blood 1987; 70(2):589-92).Clearly, one did not depart beyond this benchmark. In fact, no method isknown from the literature by which viruses or bacteria in complete PCscan be decontaminated exclusively by irradiation with UV light (i.e. UVBor UVC).

PCs are obtained by mechanical thrombapheresis from individual donors orelse are isolated from blood donations, with the platelets from severalblood donations (generally 4-6) being pooled. The volume of the PC whichis able to be obtained accordingly generally lies between approximately200 and 350 ml. However, PCs are also produced from individual blooddonations, the volume of which is correspondingly smaller (betweenapproximately 40 and 80 ml). Both in pool PCs and also in apheresis PCs,the platelets are either suspended in blood plasma or in special storagemedia with a residual plasma content of approximately 30 to 40%. The PCsare stored in flat gas-permeable plastic bags at 20-24° C.

It would be desirable to sterilise PCs in such bags with UV light.Indeed, the problem which has been mentioned exists here, namely thatthe preparations are almost impenetrable by UV light. This is to beclarified by the following calculation example: if UVB is provided forsterilisation and one assumes a PC volume of approximately 300 ml, inaddition a penetration depth of the UVB radiation of 1 mm and exposureof both sides of the bag, a suitable exposure bag would have to have asurface of at least 1500 cm².

It appears to be difficult, if not precluded, to routinely processlarger quantities of bags having such dimensions. The problem becomeseven significantly greater if, instead of with UVB, one wishes tosterilise the PCs with UVC, because its penetration depth is much less.

Surprisingly, it was found that the above problem is solved by a methodaccording to Claim 1. Preferred embodiments are the subject matter ofthe dependent claims or are described below.

According to the present invention, the PCs are moved in a suitablemanner in their exposure bags. The movement takes place here sovigorously that layers form in areas inside the PCs, these layers beingso thin that they can be penetrated by the UV radiation. At the sametime, the movement must be such that the PC suspensions are efficientlymixed in the bags. Both are to be realized when the following conditionsexist:

-   -   1. The exposure bags are highly flexible and they are not fixed        during the exposure, e.g. clamped between quartz plates. They        therefore adapt themselves to every change in form of the PC        suspension, which occurs when the bags are moved.    -   2. The movement of the bags takes place either horizontally        (linearly in a back-and-forth direction or in circular or        ellipsoid shape) or else vertically (rocked).    -   3. The exposure bags are filled to a maximum of 30%, in        particular to a maximum of 20% of their maximum filling volume.

In all cases, the reversal of the direction of movement is to be soabrupt that the majority of the PC suspension moves further into theoriginal direction, as a result of its inertia, and therefore theresidue which remains behind can form a thin layer which is penetrableby the UV radiation. In connection with the constant intermixing due tothe movement of the bags during irradiation, finally the entire PC (andthe viruses and/or bacteria contained therein) is exposed to the UVradiation. The PCs are thereby sterilised.

The exposure bags are made from UV-transparent plastic material.Suitable plastics are, for example, ethylene vinyl acetate andpolyolefins with sheet thicknesses of 1 mm and less, in particularlysheet thickness less than 0.5 mm. The exposure bags are constructed soas to be flat and preferably have no absorption maxima in the range of200 to 320 nm. In the horizontal filled state, the exposure bags areonly a few mm thick, e.g. less than 10 mm and in particular less than 5mm, preferably even less than 3 mm and they are intended to holdspecimen volumes of for example up to 200 or up to 300 ml. The maximumholding capacity (volume) of the exposure bag is, however, greater by atleast factor 3, generally by at least 5 times, preferably at least 10 oreven at least 20 times greater than the actual specimen volume containedin it, which is to be treated.

EXPERIMENTAL INVESTIGATIONS

The described experiments illustrate the efficacy of the method and arenot limited to the inactivation of the undermentioned bacteria and/orviruses. There is also no limitation to “random donor” PCs, which wereused in the described experiments, and the method according to theinvention is also able to be used on thrombapheresis preparations. Allthe experiments were carried out three to six times. The indicatedresults represent in each case the mean values±standard deviation.

Platelet Concentrates

The PCs were produced from pools of respectively 5 buffy coats, which inturn originated from regular blood donations. The PCs had a volume ofapproximately 300 to 350 ml; the platelet concentration wasapproximately 10⁹/ml. The platelets were suspended in storage mediumSSP+(product of the company MacoPharma). The residual plasma content wasapproximately 30 to 40%.

Bacteriological Investigations

The following strains of bacteria were used in the inactivationexperiments:

-   -   Staphylococcus (S.) epidermis    -   Staphylococcus (S.) aureus    -   Bacillus (B.) cereus    -   Klebsiella (K.) pneumoniae.

Concentrations of bacteria were determined by means of a colonyformation assay and are expressed as colony forming units (CFU)/ml. Inthe experiments for inactivation of bacteria, complete PCs or PCaliquots were spiked with 10⁴ to 10⁵ CFU/ml of one of the indicatedspecies and then irradiated with UV light.

Virological Investigations

PC aliquots were spiked with suid herpes virus (SHV-1, pseudorabiesvirus, Aujeszky strain) or vesicular stomatitis virus (VSV, Indianastrain). Virus titers were determined by means of CPE assay(CPE=cytopathic effect). They are indicated as TCID₅₀ (TCID=tissueculture infective dose). Vero cells served as indicator cells. Theinitial virus concentration in the experiments which were carried outwas approximately 10⁵ to 10⁷ TCID₅₀.

Exposure Apparatus

One of the two exposure apparatus used was equipped with tubes whichemitted UVB light. The irradiation took place from both sides of theexposure bags which were put in place, i.e. from above and from below.The exposure apparatus was provided with a shaking device which carriedout back-and-forth movements at a frequency of 60 changes ofdirection/min. A second exposure apparatus was likewise equipped withtubes which emitted UVB light. The irradiation likewise took place fromboth sides. A third apparatus (of the same type of construction as thesecond) was provided with tubes which emitted UVC light (wavelength: 254nm). Both apparatus were able to be provided with 2 different shakingdevices: one horizontal shaker, which carried out ellipsoidback-and-forth movements, and a rocking shaker.

Exposure Bags

The exposure bags which were used consisted of ethylene vinyl acetate(EVA), which is penetrable by UV light. Two sizes of bags were used:

-   -   1. 14.5×18.5 cm (external bag area approx. 268 cm²)    -   2. 22.5×38 cm (external bag area approx. 855 cm²)

In the experiments with the small EVA bags, the sample volume was 80 ml,in those with the large bags approximately 300-350 ml (complete PCs weretreated).

Experiment Example 1 Inactivation of S. epidermidis by UVB, with andwithout Free Movement of the Platelet Suspension During Shaking

In the experiment, the sample volume was 80 ml. The free mobility of theplatelet suspension during shaking, and hence the formation of a thinlayer was prevented in a sample in that the exposure bags were clampedsecurely between two quartz plates. The resulting layer thickness wasapproximately 3 mm. In the second sample, the distance between thequartz plates was increased such that the platelet suspension waslargely able to move freely during shaking. The two samples wereirradiated with 1 J/cm².

As Table 1 shows, the bacteria titer in the fixed samples was reduced byapproximately 2 log₁₀, but by more than 4 log₁₀ in the loosely placedones.

TABLE 1 Sample title Bacteria titer(log₁₀ CFU/ml) Untreated control 4.1± 0.03 Securely clamped sample 1.4 ± 1.29 Loosely placed sample −0.40 ±0.35 

Experiment Example 2 Inactivation of S. epidermidis by UVB in CompletePCs, Loosely or Securely Clamped Exposure Bags, with Shaking

The PC volume in this experiment was 330 ml, the average layer thicknessin large EVA bags was accordingly approximately 3.9 mm. The PCs wereirradiated with 3 UVB doses (0.8, 1.0 and 1.2 J/cm²) under the followingconditions:

-   -   1. without shaking, loosely placed between quartz plates    -   2. with shaking, pressed between quartz plates.

As the experiment results show (Table 2), in the PCs which were securelyclamped during shaking, the bacteria titer was reduced by the UVBtreatment by up to approximately 2 log₁₀, by comparison in the looselyplaced samples, depending on the dose, by approximately 3.4 to more than4 log₁₀.

TABLE 2 Bacteria Titer Sample placement UVB Dose(J/cm²) (log₁₀ CFU/ml)shaken, securely clamped 0 4.11 ± 0.00 shaken, securely clamped 0.8 2.14± 0.48 shaken, securely clamped 1.0 1.95 ± 0.03 shaken, securely clamped1.2 1.99 ± 0.03 shaken, loose 0 4.19 ± 0.11 shaken, loose 0.8 0.77 ±0.69 shaken, loose 1.0 −0.14 ± 0.05  shaken, loose 1.2 −0.40 ± 0.05 

Experiment Example 3 Inactivation of Further Bacteria in Freely Movableor Fixed PC Aliquots by UVB

It can be seen from the first two experiment examples that S.epidermidis is effectively inactivated in PCs under the condition thatthe PC suspension can move freely during the UV irradiation. In thefollowing experiment, the following additional strains of bacteria weretested: S. aureus, B. cereus and K. pneumoniae.

The conditions were the same as described in Experiment Example 1. Inall three cases a similar result as with S. epidermidis: in the looselyplaced PC samples, the bacteria were inactivated by approximately 3.9 to4.29 log₁₀, whereas the titers in the fixed samples were only reduced byapproximately 2 to 3.4 log₁₀ (Table 3).

TABLE 3 Strain of Bacteria Titer bacteria Sample Title (log₁₀ CFU/ml) S.aureus untreated control 4.88 ± 0.00 S. aureus securely clamped, shaken1.49 ± 1.30 S. aureus loosely placed, shaken 0.97 ± 0.86 B. cereusuntreated control 4.99 ± 0.09 B. cereus securely clamped, shaken 2.99 ±0.13 B. cereus loosely placed, shaken 0.74 ± 0.68 K. pneumoniaeuntreated control 4.94 ± 0.08 K. pneumoniae securely clamped, shaken2.34 ± 0.24 K. pneumoniae loosely placed, shaken 1.00 ± 0.89

Experiment Example 4 Inactivation of S. epidermidis in PC Aliquots byUVB Under Various Shaking Conditions

An investigation was carried out as to whether the inactivation of S.epidermidis in loosely placed PC aliquots is also increased whendifferent horizontal shakers are used from the one used in Experiments 1to 3, which, as mentioned, makes back-and-forth movements. In thefollowing experiments, an orbital shaker was used which carried out acircular movement (radius: 3 cm, rotation rate: 50/min), in addition arocker with 50 up- and down movements per minute. Again, one of the twosamples (80 ml) was clamped securely between quartz plates, the otherwas placed loosely. As can be seen from the results shown in Table 4,this time the extent of the bacteria inactivation was higher by 3 to 4log₁₀ in the loosely placed PC samples than in the securely clampedones.

TABLE 4 Bacteria Titer Shaker Sample Title (log₁₀ CFU/ml) — untreatedcontrol 4.94 ± 0.16 Orbital securely clamped 4.23 ± 0.00 Orbital looselyplaced 0.50 ± 0.39 Rocker securely clamped 4.02 ± 0.17 Rocker looselyplaced 0.87 ± 0.78

Experiment Example 5 Inactivation of Suid Herpes Viruses by UVB, withoutor with Free Movement of the PC Aliquots During Shaking

In order to test whether the increase of inactivation of pathogens inPCs which are not fixed during irradiation with UV light, relates notonly to bacteria but also to viruses, the following experiment wascarried out: PC aliquots of 80 ml were spiked with suid herpes viruses(SHV-1) and were treated with UVB as described in Experiment Example 1.In the freely placed samples, the virus titer was reduced by almost 4log₁₀, in the securely clamped samples, on the other hand, only byapproximately 3 log₁₀. This confirms that under the said conditions, theinactivation of viruses is also distinctly improved.

TABLE 5 Sample Title Virus Titer (Log₁₀ TCID₅₀) untreated control 4.4 ±0.2 securely clamped sample 1.41 ± 0.18 loosely placed sample 0.56 ±0.15

Experiment Example 6 Inactivation of Vesicular Stomatitis Viruses byUVB, without or with Free Movement of the PC Aliquots During Shaking

The experiment was carried out as described in Experiment Example 5,except that the PC aliquots were spiked with VSV instead of with SHV-1.Again, the extent of the virus inactivation in the loosely placedsamples was greater by more than 6.46 log₁₀ than in the comparativesamples, which were fixed between quartz plates during exposure (Table6). It is, in fact, noticeable that also in these, the virus titer wasrelatively greatly reduced (by approximately 5.4 log₁₀). VSV is clearlymore UV-sensitive than SHV-1.

TABLE 6 Sample Title Virus Titer (Log₁₀ TCID₅₀) untreated control  6.7 ±1.05 securely clamped sample 1.29 ± 1.05 loosely placed sample ≦0.24 ±0.00 

Experiment Example 7 Inactivation of S. epidermidis by UVC, without orwith Free Movement of the PC Aliquots During Shaking

In this experiment, the irradiation took place with UVC instead of UVB.The UV dose was 0.3 J/cm² (exposure time: 60 sec). Otherwise, theconditions were as described in Experiment Example 1. As can be seenfrom Table 7, the bacteria titer in the fixed samples was only reducedby approximately 1 log₁₀. This clearly reflects the small penetrationdepth of UVC into the platelet suspension. In the loosely placedsamples, on the other hand, no more bacteria were able to be found; theinactivation factor was greater than 4 log₁₀.

TABLE 7 Sample Title Bacteria Titer (log₁₀ CFU/ml) untreated control4.08 ± 0.04 securely clamped sample 2.99 ± 0.13 loosely placed sample≦0.24

Experiment Example 8 Inactivation of VSV by UVC, without or with FreeMovement of the PC Aliquots During Shaking

As in Experiment Example 6, VSV was used as the test virus. Theconditions were the same as in Experiment Example 7. The results shownin Table 8 show that also in this case, the extent of pathogeninactivation was much more marked in the loosely placed samples than inthe fixed ones: whereas the virus titer in the latter was only reducedby approximately 1.5 log₁₀ it was approximately 6.2 log₁₀ in thenon-fixed samples.

TABLE 8 Sample Title Virus Titer (Log₁₀ TCID₅₀) untreated control 6.92 ±0.22 securely clamped sample 5.47 ± 0.21 loosely placed sample 0.74 ±0.76

1. A method for inactivating pathogens and/or leucocytes in plateletconcentrates (PCs) comprising the steps: providing PCs obtained fromdonor blood and/or by mechanical apheresis, exposing the PCs toirradiation with ultraviolet (UV) light, in which the PCs consist of aplurality of units which are able to be handled individually and storedseparately, and the PC units are each placed in flexible, UV-penetrableflat exposure bags, characterized in that the exposure bags are filledto less than 30% of the maximum filling volume of the exposure bags andthe exposure bags are moved during the irradiation with UV light so thatthe contents of the exposure bags are circulated and zones of changinglayer thicknesses are formed by the movement.
 2. The method according toclaim 1, characterized in that the pathogens are viruses and/orbacteria.
 3. The method according to claim 1, characterized in thatthrough the movement, the zones have irradiated regions for whichregularly at times layer thicknesses below 1 mm are given.
 4. The methodaccording to claim 1, characterized in that the movement, in particularthe amplitude of the movement, takes place so that within the PCsirradiated regions are formed in which the layer thickness is regularlyat times less than 0.5 mm.
 5. The method according to claim 1,characterized in that the exposure bags have an underside and an upperside and the sum of the areas of the underside and the upper side, whichis or can be in contact with the bag content, is more than 90 areapercent, preferably more than 99 area percent, of the inner totalsurface of the bag content.
 6. The method according to claim 1,characterized in that the UVB irradiation is or comprises less than 320to 280 nm and/or UVC less than 280 nm to 200 nm, in particular UVC lessthan 280 to 200 nm, and preferably consists exclusively of irradiationwith wavelengths of the above-mentioned ranges.
 7. The method accordingto claim 1, characterized in that each unit is produced from samples upto a maximum of 8 donors, preferably of up to a maximum of 6 donors. 8.The method according to claim 1, characterized in that each unitoriginates from one donor.
 9. The method according to claim 1,characterized in that the PC contains at least 1×10⁸ platelets per ml,preferably at least 5×10⁸ platelets per ml.
 10. The method according toclaim 1, characterized in that the irradiation with UVB takes place witha light energy of 0.3 to 5 J/cm², preferably from 0.5 to 2.5 J/cm². 11.The method according to claim 1, characterized in that the irradiationwith UVC takes place with a light energy of 0.01 to 2 J/cm², preferablyof 0.1 to 1 J/cm².
 12. The method according to claim 1, characterized inthat the PCs contain plasma and if applicable a suitable storage medium,in which the plasma content is preferably greater than 20% by weight.13. The method according to claim 1, characterized in that the PCscontain a buffered aqueous storage medium.
 14. The method according toclaim 1, characterized in that viruses, bacteria and/or leucocytes areinactivated and the function of the platelets remains substantiallyunchanged.
 15. The method according to claim 1, characterized in thatthe exposure bags have a volume of up to 5000 ml.
 16. The methodaccording to claim 1, characterized in that the exposure bags are heldso as to be movable in the apparatus in which they are moved andirradiated, and in particular are not clamped between two surfaces, e.g.UV-penetrable glass or plastic plates.
 17. The method according to claim1, characterized in that the exposure bags are moved during at leastthree quarters of the total exposure duration.
 18. The method accordingto claim 1, characterized in that the exposure bags are moved byshaking.
 19. The method according to claim 1, characterized in that theexposure bags are moved by rocking.
 20. The method according to claim 1,characterized in that the exposure bags are moved by rotation.
 21. Themethod according to claim 1, characterized in that the shaking takesplace with an orbital shaker, platform shaker, rocker shaker or tumblershaker.
 22. The method according to claim 1, characterized in that theexposure bags are placed on one side so that during and by the movementor shaking, the height of the exposure bag, viewed in relation to thedistance along the surface normal between the surface on which theexposure bags lie, and the point of intersection with the upper surfaceof the exposure bag over the entire upper surface of the exposure bagwhich is in contact with the bag contents changes constantly.
 23. Themethod according to claim 1, characterized in that the exposure bagshave an average filling level of 10, preferably of less than 5 mm, andthrough the movement wave troughs are constantly produced which havelayer thicknesses of less than half of the mean filling level,preferably layer thicknesses of less than 1 mm or even less than 0.1 mm.24. The method according to claim 1, characterized in that during theexposure, the exposure bags are moved constantly with an amplitude of0.2 to 8 cm at least in the x-direction and if applicable also in they-direction (y-direction at right-angles to the x-direction) andindependently therefrom the frequency of the change in direction of theshaking movement is 0.5 to 10 Hz.
 25. The method according to claim 1,characterized in that during the exposure, the exposure bags are filledto a maximum of 20% of their maximum filling volume.